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Original Article | Artículo Original
Selective cytotoxic effect of 1-O-undecylglycerol in human melanoma cells
[Efecto citotóxico selectivo del 1-O-undecilglicerol en células de melanoma humano]
Marian Hernández-Colina1*, Alberto Martín Cermeño1, Alexis Díaz García2
1Instituto de Farmacia y Alimentos, Universidad de la Habana, Calle 222, No. 2317, entre 23 y 31, La Coronela, La Lisa, CP 13600, La Habana, Cuba. 2
Laboratorios de Producciones Biofarmacéuticas y Químicas (LABIOFAM). Ave. Boyeros Km 16½, Rpto. Mulgoba, Boyeros, La Habana, Cuba. *E-mail: marianhc@ifal.uh.cu
Abstract Resumen
Context: 1-O-alkylglycerols are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed antineoplastic activity for this family of compounds, structurally related to alkylphospholipids, but the activity of linear chain synthetic alkylglycerols in cancer cell lines is less documented. Melanoma is a high incidence cancer, highly resistant to potential treatments. Finding new anti-cancer compounds to improve melanoma prognosis is a relevant research issue.
Aims: To study the cytotoxic effect of 1-O-undecylglycerol in primary cultured normal fibroblasts and A375 human melanoma cell line.
Methods: Cells were treated with different concentrations of 1-O-undecylglycerol and viability assessed by MTT assay. Morphological changes were visualized by DAPI and acridine orange-ethidium bromide staining. Mitochondrial membrane potential was evaluated, and gene expression of P53 and BcL-2 was semi-quantified.
Results: 1-O-undecylglycerol decreased viability of A375 cells and exerted very low cytotoxicity on primary cultured normal fibroblasts. Necrosis appeared in A375 cells but not in fibroblasts, and no apoptotic changes were visualized in DAPI staining experiments. After 24 h fibroblasts and melanoma cells developed mitochondrial potential changes similar to valinomycin. The gene expression of P53 and BcL-2 decreased in treated cells.
Conclusions: 1-O-undecylglycerol exhibited selective cytotoxic activity in A375 melanoma cells when compared with primary cultured fibroblast. Its toxicity is mediated by necrosis that may be related with mitochondrial events and decrease in P53 and BcL-2 expression. The results suggest that UDG could be a useful strategy to combine with other chemotherapeutic agents in melanoma treatment.
Contexto: Los 1-O-alquilgliceroles son éteres presentes en el aceite de hígado de tiburón y en la leche materna. Han mostrado actividad antine-oplásica, pero está poco documentada para alquilgliceroles sintéticos de cadena lineal. La búsqueda de nuevos antitumorales para mejorar el pronóstico del melanoma resulta relevante, debido a su alta incidencia y resistencia.
Objetivos: Estudiar el efecto citotóxico del 1-O-undecilglicerol en cultivo primario de fibroblastos normales y la línea celular A375 de melanoma humano.
Métodos: En células tratadas con diferentes concentraciones de 1-O-undecilglicerol se monitoreó la viabilidad mediante ensayo con MTT. Los cambios morfológicos se visualizaron por tinción con DAPI y naranja acridina- bromuro de etidio. Se evaluó el potencial de membrana mito-condrial, y se semi-cuantificó la expresión de los genes P53 y BcL-2.
Resultados: El 1-O-undecilglicerol redujo la viabilidad de las células A375, y ejerció poca citotoxicidad en el cultivo primario de fibroblastos norma-les. Ocurrió necrosis en las células A375 pero no en los fibroblastos, y no se visualizaron cambios apoptóticos en la tinción con DAPI. Luego de 24h ambas células desarrollaron cambios en el potencial de membrana mito-condrial similar a la valinomicina, y disminuyó la expresión de los genes p53 y BcL-2.
Conclusiones: El 1-O-undecilglicerol mostró actividad citotóxica selectiva en células A375 al compararlo con cultivo primario de fibroblastos huma-nos. Su toxicidad es mediada por necrosis, probablemente por eventos mitocondriales y disminución en la expresión de P53 y BcL-2. Ello sugiere que el 1-O-undecilglicerol pudiera ser útil en combinación con otros qui-mioterapéuticos en el tratamiento del melanoma.
Keywords: Alkylglycerols, cytotoxicity, melanoma, primary cultured fi-broblasts.
Palabras Clave: Alquilgliceroles, citotoxicidad, cultivo primario de fibroblastos melanoma, necrosis.
ARTICLE INFO
Received | Recibido: February 5, 2016.
Received in revised form | Recibido en forma corregida: April 5, 2016. Accepted | Aceptado: April 5, 2016.
Available Online | Publicado en Línea: April 9, 2016.
Declaration of interests | Declaración de Intereses: The authors declare no conflict of interest. Funding | Financiación: The authors confirm that the project has not funding or grants. Academic Editor | Editor Académico: Gabino Garrido.
INTRODUCTION
Shark liver oil (SLO) is successfully used as therapy for several diseases, i.e. cancer, inflamma-tion (Iannitti and Palmieri, 2010). Obtained from sharks that live in deep cold waters, it contains alkylgly-cerols (AKG) and squalene as major components, among other lipids as unsaturated fatty acids (Bordier et al., 1996, Qian et al., 2014). SLO exhibited antineoplastic activity in Walker 256 tumor-bearing rats (Iagher et al., 2013), and in human mam-mary, ovarian carcinoma and prostate cancer cell lines (Krotkiewski et al., 2003). This activity is related to AKG content, and SLO with low content of AKG showed no activity in transformed cells (Davidson et al., 2007).
AKG are alkyl ethers of glycerol that represent about 20% of SLO (Hallgren and Larson, 1962), usually as follows: 12:0 O-dodecylglycerol), 1-2%; 14:0 tetradecylglycerol), 1-3%; 16:0 (1-O-hexadecylglycerol, chimyl alcohol), 9-13%; 16:1 n-7 hexadec-7-enylglycerol), 11-13%; 18:0 O-octadecylglycerol, batyl alcohol), 1-5%; 18:1 n-9 (1-O-octadec-9-enylglycerol, selachyl alcohol), 54-68%; 18:1 n-7 (1-O-octadec-7-enylglycerol), 4-6%, and other (< 1%). This family of compounds acts as biological response modifiers. As anti-tumor agents, they have been related to inhibitory growth, antimetastatic activity, anti-neoangiogenic action, and induce differentiation and apoptosis in cancer cells (Deniau et al., 2011, Molina et al., 2013). Im-mune stimulation properties (Kantah et al., 2012; Qian
et al., 2014)and brain drug delivery enhancing (Hülper
et al., 2013) have also been attributed to these
sub-stances. The antineoplastic actions of linear chain synthetic AKG in cancer cell lines is less docu-mented, but Sotomayor demonstrated cytotoxic activity in MCF-7 breast cancer cells (Sotomayor et al., 2006).
Non-selective cytotoxicity and drug resistance are recurrent issues in most of available cancer chemotherapy. In this context, natural products, derived from plants or animals, have drawn atten-tions of many scientists (Block et al., 2015).
Melanoma is a malignant skin tumor induced by transformation of melanocytes, whose incidence rate is rapidly increasing in the world. Metastatic melanoma has a very poor prognosis, a fact that is
in part due to its high resistance to cytotoxic agents (Cattaneo et al., 2015). Hence, finding new anti-cancer compounds, to improve melanoma progno-sis is a relevant research issue. In melanoma, im-mune regulation at the level of the tumor microen-vironment is a key factor in treatment (Galon et al., 2012), so immunomodulating agents as AKG could be a right choice. The combination of immunomodulatory action and antineoplastic po-tential could be a good strategy in melanoma treatment. It becomes relevant to study the cell death mechanisms associated with AKG cytotoxici-ty in melanoma cells.
In this context, the aim of the present study is to evaluate the cytotoxic properties of synthetic 1-O-undecylglycerol (UDG) in primary cultured normal fibroblasts and A375 human melanoma cells. It was further studied the cell death induced by UDG, its relationship with gene expression of death associated proteins, and changes in mito-chondrial potential.
MATERIAL AND METHODS
Compound and reagents
All reagents were obtained from Sigma–Aldrich Corp. (St. Louis, MO, USA), unless otherwise stat-ed. 1-O-undecylglycerol was synthesized in Food and Pharmacy Institute, with 95% of purity, by Williamson´s synthesis, as previously reported (Le-ón et al., 2002). A stock solution of UDG was prepared in absolute ethanol at 50 mM. In every experiment, it was diluted in complete culture media to 150 µM as work solution.
Primary culture of human fibroblasts (FF) and A375 human melanoma cells
fi-broblast were cultured in Dulbecco’s Modified Ea-gle’s medium (DMEM) with 10% fetal bovine se-rum, 100 U/mL penicillin and 100 μg/mL strept o-mycin, in T-25 cm2 tissue culture flask (Corning Costar Co., Lowell, MA, USA), and maintained in an incubator with a humid atmosphere at 37°C un-der 5% CO2.
A375 cells were obtained from the American Type Culture Collection (ATCC CRL-1619). The cell line was cultured in RPMI-1640 (Hyclone, UT, USA), supplemented with 10% fetal bovine serum (Gibco, Life Technologies, USA) and 50 µg/mL gen-tamycin, in T-25 cm2 tissue culture flasks. Cells were passaged twice a week at the ratio of 1:5 to keep an exponentially growing state.
Cell viability assay
The viabilities of A375 cells or primary cultured fibroblasts were evaluated using the [3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide] (MTT) colorimetric assay (Mossman, 1983). Both types of cells (6 x 103) were incubated for 24 or 48 h in 96-well microtiter cell culture plates, in the absence (control cells) or presence of UDG, in a 200 µL final volume. After the treatments, cells
were incubated for 4 h at 37⁰C with 20 µL of MTT 5
mg/mL. The blue MTT formazan precipitate was dissolved in 50 µL of isopropyl alcohol, and after 30
minutes of agitation at 37⁰C, the absorbance was
measured at 570 nm on a multiwell plate reader SUMA PR-521 (TecnoSuma, Cuba). Cell viability is expressed as a percentage of these values in treated cells, compared to the non-treated (control) cells.
Morphological changes visualization by DAPI and acridine orange-ethidium bromide (AO/EtBr) staining
For visualization of morphological changes, A375 or FF cells (4 x 104) were cultured in 24 wells plate, and treated with low concentrations of UDG for 24 or 48 h, or untreated. FF cells were treated with 18.75 µM UDG, correspondent to 1/8 of maxi-mum concentration assayed. A375 cells were treat-ed with 5 µM UDG, which represents 1/16 of con-centration that inhibits 50% control viability after the incubation period (IC50). Fixation solution was added, 500 µL of acetic acid/methanol (1:2) for 2 min, and then plates were washed twice.
6-diamidino-2-phenylindole (DAPI) staining (1
mg/mL) was added for 20 minutes, at 20⁰C in the
dark.
For AO/EtBr assay, cells were stained with a 1:1 mix of 100 µg/mL acridine orange and ethidium bromide in 1x PBS solution for 5 minutes. In both cases, cells were then observed under a fluores-cence microscope (Olympus IX71, camera Olympus DP72, Japan) at 10x magnification.
Mitochondrial membrane potential
The mitochondrial membrane potential was visualized using Mitochondria Staining Kit (for mi-tochondrial potential changes detection; Sigma, St. Louis, MO, USA), which includes JC-1 dye. The JC-1 probe is a cationic dye that exhibits membrane po-tential-dependent accumulation in mitochondria, as indicated by a fluorescence emission shift from green to red. Mitochondrial depolarization is spe-cifically indicated by a decrease in the red-to-green fluorescence intensity ratio (Cossarizza et al., 1993). The cells were incubated for 1, 12 or 24 h in the ab-sence (control) or preab-sence of UDG (18.75 µM in FF cells, and 5.25 µM for A375 cells) or valinomycin 1 µg/mL. After the JC-1 dying, fresh media was add-ed, and observed under a fluorescence microscope (Olympus IX-71, Japan), with 480 nm and 520 nm filters. Fields were photographed with an Olympus DP-72 camera (Olympus, Japan).
Reverse transcription polymerase chain reaction (RT-PCR)
Total cellular RNA was extracted from treated and control cells, A375 and FF, at 24 and 48 h after treatment, using 1 mL Trizol reagent (Invitrogen, USA) according to the manufacturer’s instruction.
The extracted RNA was dissolved in RNase-free water. The RNA concentration was determined us-ing spectrophotometer (Bio Photometer plus, Eppendorf). Equivalent volume for 2 µg of RNA was reverse transcribed using 2 µL of reverse tran-scriptase (RT). RT was carried out for 60 min at
42⁰C in a thermocycler (Nashita 4196, Spain). PCR
was performed in a total reaction volume of 25 µL
that contained 5 µL of cDNA solution and 1.25 μL
of sense and antisense primers. The primers for GAPDH, p53 and Bcl-2 in forward and reverse were
CCTTCCTGGGCATGGAGTCCTG-3’ and GGAGCAATGAGCTTGATCTC-3’; p53 (100 bp): GGGTTAGTTTACAATCAGCCACATT-3’ and 5’-GGCCTTGAAGTTAGAGAAAATTCA-3’; Bcl-2 (100 bp): 5’-CAGGTCCTCTCAGAGGCAGATAC-3’ and 5’-CCTCTCCAGGGACCTTAACG-3’.
PCR was performed using the following proce-dure: 59⁰C for 1 min followed 35 cycles of 94⁰C for
1.5 min and 72⁰C for 1 min, and completed with one cycle at 72⁰C for 7 min. For visualization, PCR
products were analyzed on a 2% agarose gel. The gel images and relative intensity of each band were obtained using Quantity one software (Bio-Rad, USA), after normalized to the corresponding GAPDH band.
Statistical analysis
The software program GraphPad Prism 5.0 (GraphPad Software Inc., USA) was used for statis-tical analysis. The data are expressed as mean ± standard error of mean (SEM). Comparisons be-tween the different groups were performed by the one-way analysis of variance (ANOVA) followed by the Newman–Keuls multiple comparison test. Dif-ferences at p < 0.05 were considered statistically significant. The IC50 values for cell viability were estimated using a non-linear regression algorithm.
RESULTS
Cell viability assay
Incubation of A375 cells with UDG (Fig. 1A-B) promoted significant decrease in cell viability in concentration and time-dependent manner, as es-timated by the MTT assay. The assessed IC50 value was 94.59 µM for 24 h and 82.06 µM for 48h. FF cells viability was slightly affected by UDG treat-ment for 24 h in a concentration-dependent man-ner. The IC50 for these cells was not reached for assayed concentrations. At 48 h (Fig. 1B), FF cells keep their viability, and A375 cells are more affect-ed by UDG from 75 µM. Accordingly, these results reveal that UDG exhibited more selectively activity against melanoma cells, with low cytotoxicity to normal fibroblasts.
Morphological changes of FF and A375 cells by AO/EtBr and DAPI staining
Cells were stained with AO/EtBr to visualize cell death after 24 and 48 h of treatment. Morphologi-cal changes in A375 and FF cells were observed un-der the fluorescence microscope after exposure to UDG and shown in Fig. 2. After 24 h both cell lines were affected by UDG, and almost all cells exhibit-ed orange-rexhibit-ed fluorescence, indicating increasexhibit-ed membrane disruption; and looked intact, with nu-clear morphology resembling that of viable cells, which appears to be a necrotic event. At 48 h A375 cells showed clear growth inhibition, with an or-ange nucleus, determining necrotic events. FF cells exhibited yellow nucleus, as a signal of recovery of their viability. The absence of DNA fragmentation was confirmed by DAPI staining. Neither A375 nor FF cells showed apoptotic characteristics when stained with DAPI.
Changes in mitochondrial membrane potential
To explore the effects of UDG on the mitochon-drial membrane potential, FF and A375 cells were cultured with UDG or valinomycin for 1, 12 and 24 h, and then processed for confocal microscopy analysis after being stained with fluorescent probe JC-1. The dye enters selectively into the mitochon-dria and reversibly changes its color from red to green as the membrane potential decreases (Cossarizza et al., 1993). After treatment with UDG, the cells showed increased green fluorescence similar to valinomycin as a positive control, in a time-dependent manner (Fig. 3). It suggests that UDG disrupts the mitochondrial membrane potential, resulting in the cytosolic accumulation of mono-meric JC-1.
p53 and BcL-2 gene expression using RT-PCR
A
B
Figure 1. Effects of 1-O-undecylglycerol (UDG) on A375 cells and normal fibroblasts (FF) via-bilities, estimated by the MTT assay. Panels (A) and (B) display the effects at 24 and 48 h, re-spectively.
Cont rol U DG 2 4 h U DG 4 8 h
A
FF
B
C
A3 7 5
D
Figure 2. Effects of 1-O-undecylglycerol (UDG) on morphological changes in fibroblasts (FF) and A375 cells, detected by AO/EB double fluorescence (green-orange) and DAPI staining (blue).
FF and A375 cells were incubated with UDG 18.75 µM and 5 µM, respectively for 24 or 48 h. (A) FF cells stained with AO/EB and (B) with DAPI. (C) A375 cells stained with AO/EB and (D) with DAPI.
Cont rol 1 h 1 2 h 2 4 h
FF
V a linom yc in
U DG
A3 7 5
V a linom yc in
U DG
Figure 3. Effects of 1-O-undecylglycerol (UDG) and valinomycin on mitochondrial membrane potential changes in fibroblasts (FF) and A375 cells, detected by JC-1 staining.
A
B
Figure 4. Semi-quantitative RT-PCR analysis of p53 and BcL-2 gene expression in fibroblasts (A) and A375 (B) cells, control (C) and treated with 1-O-undecylglycerol (UDG) for 24 or 48 h, with GAPDH as housekeeping gene. FF and A375 cells were incubated with UDG 18.75 µM and 5 µM, respectively.
The bars are mean ± SEM of three experiments. *p<0.05, ** p<0.01 respect to the control as measured by 1-way ANOVA.
DISCUSSION
Melanomas cause over 76% of skin cancer deaths annually. Their incidence continues to rise at an alarming rate (Siegel et al., 2015). They are among the most resistance cancers to chemothera-peutic agents. Thus, there is an urgent need to
ex-plore other alternative and targeted therapies (Hutchinson, 2015). Alkyl-ether lipids from SLO have shown selective effects in inhibiting the prolifera-tion of neoplastic cells, with practically no effects on normal cells (Krotkiewski et al., 2003), similar to
p53
bcl-2
gapdh
C 24 h 48 h
p53
bcl-2
gapdh
their alkylphospholipid synthetic analogues (van Blitterswijk and Verheij, 2013).
In the present study, the cytotoxic effects of UDG, a synthetic AKG, were analyzed in primary cultured human fibroblasts FF and A375 human melanoma cells. UDG exhibited selective antiproliferative activity on A375 cells, and FF cells were resistant to the treatment after 48 h, in a con-centration-dependent manner (Fig. 1A-B).
Although AKG have been previously studied for anticancer properties (Ianniti and Palmieri, 2010; Deniau et al., 2011), such activity by UDG is here reported for the first time. The induction of apoptosis and/or necrosis by shark liver oil has been attributed to the presence of AKG on its composition (Krotkiewski et al., 2003), but UDG has not been reported as a usual component of that oil.
For a long time, necrosis has been considered as a merely accidental cell death mechanism and was defined by the absence of morphological traits of apoptosis or autophagy, but now is clear that ne-crotic cell death has a prominent role in multiple physiological and pathological settings (Galluzzi et al., 2012).
Multidrug-resistant tumors remain susceptible to necrotic death (Sasi et al., 2009). In this way, necro-sis should become a focus of study in determining novel approaches to cancer prevention and treat-ment. To confirm if necrosis was present in UDG induced cell death, cells were stained with AO, which permeates all cells and the nuclei become green, and EtBr, which is only taken up when cyto-plasmic membrane integrity is lost, and their nu-clei are stained orange/red (Kasibhatla et al., 2006). UDG induced morphological changes consistent with necrotic events in both cell lines, but FF cells required higher concentration, and seems to get recovered, as shown in Fig. 2, where necrotic char-acteristics appeared in treated cells, and non-apoptotic features were detected by DAPI staining.
The primary molecular mechanism behind ne-crosis is believed to be the rapid depolarization of membranes leading to swelling and rupture of the plasma membrane (Lemasters et al., 1998), and AKG can incorporate into membrane phospholipids and modify lipid structure of membranes (Marigny et al., 2002) as part of their mechanism of cell death in-duction.
A hallmark of necrotic cell death is mitochon-drial dysfunction itself, such that if mitochonmitochon-drial structure function are preserved during a necrotic insult, the cells affected typically no longer die (Crompton, 1999). A rapid decrease in mitochondrial
membrane potential (∆Ψm) was detected after 1h
of treatment with UDG, and at 24 h the effect was similar to valinomycin, used as positive control (Fig. 3). Edelfosine, an ether lipid structurally
relat-ed with AKG, also affectrelat-ed ∆Ψm about cell death
(Vrablic et al., 2001), so the glyceryl ether bond could be a factor in mitochondrial disruption induced by those compounds.
The mitochondrial loss of internal permeability
is a primary factor for the loss of ∆Ψm and aden
o-sine triphosphate (ATP) that promotes osmotic shock to the organelle. This is responsible of necro-sis, due to loss of ion homeostasis and cell integrity (Bonora and Pinton, 2014). Mitochondria could be a po-tential target of selective activity of UDG because its membrane potential was affected in both fibro-blasts and melanoma cells, but only melanoma cells exhibited significant cell death, so the relative number of mitochondria increased by Warburg effect (Kim and Dang, 2006) could be responsible for exposing these cells to necrosis induced by UDG.
Taking into account that A375 cells showed a significant decrease of cell viability in MTT assay (Fig. 1A-B), and a decrease in mitochondrial mem-brane potential, it should be extra-mitochondrial components sensitive to MTT dye. Recent research on the site of reduction of MTT has refuted the dogma that MTT is always reduced in the mito-chondria (Bernas and Dobrucki, 2002). Studies indicate that NADH is responsible for most MTT reduction and is associated not only with the mitochondria but also with the cytoplasm and associated with membranes in the endosome/lysosome compart-ment as well as the plasma membrane. MTT reduc-tion at the plasma membrane may account for ob-servations of formazan crystals occurring outside of cells.
mela-noma cells resulted in decreased proliferation (Avery-Kiejda et al., 2011), as was observed in A375 cells treated with UDG. The interference in the regula-tion of BcL-2 could be a factor in UDG necrosis in-duction although the role of BcL-2 in necrosis has not been extensively studied. It has been recog-nized as a protein that inhibits apoptosis, leading to cell survival (Tomek et al., 2012), which overexpres-sion protected cancer cells from cell death induced by ether lipid edelfosine (Gajate and Mollinedo, 2014).
CONCLUSIONS
This study is the first to demonstrate cytotoxic activity of UDG. This activity is selective in A375 melanoma cells when compared with primary cul-tured fibroblast. A particular finding is that pre-dominant cell death is necrosis, associated with mitochondrial events and decrease in P53 and BcL-2 expression. The results suggest that UDG could be a useful strategy to combine with other chemo-therapeutic agents in melanoma treatment.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENT
The authors are grateful to Instituto Pedro Kouri, La Ha-bana, Cuba, for the use of some facilities for the experiments.
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